(1) Field of the Invention
The present invention relates to an apparatus analyzing nucleic acids, and in particular, an apparatus capable of analyzing gene sequences, gene polymorphism, and gene mutation.
(2) Description of Related Art
For determining DNA base sequences, methods using gel electrophoresis and fluorescence detection are widely used. In such method, first, many copies of a DNA fragment are made, the sequence of which is to be analyzed. Fluorescence-labeled fragments of various lengths are prepared, 5′-terminals of the DNA being starting points, wherein fluorescent labels are also attached, wavelengths varying with bases at 3′-terminals of these DNA fragments. The difference in length is identified by one base by gel electrophoresis, and emission from each of the fragment groups is detected. DNA terminal base types of the DNA fragment groups being studied are elucidated according to colors of emission wavelengths. The DNA fragment groups pass through the fluorescent detection section one by one from a shorter one, so that terminal base types can be identified consecutively from a shorter DNA by measuring fluorescence colors. Thereby, the sequence is determined. Such fluorescent DNA sequencers are widespread, and also their contribution to the Human Genome Project was enormous. On the other hand, the Human Genome Project was completed, as declared in 2003, and the time has come to make use of sequence information in medicine and various industries. There, in many cases, analysis of entire long DNA is not required and elucidation of a short DNA sequence of interest is sufficient. For such DNA sequence analysis, simple methods and apparatuses are required.
The sequence determination by stepwise chemical reactions such as pyrosequencing is a technique developed in order to meet such requirement (for example, Patent document 1 and Patent document 2). In this method, a primer is hybridized with a target DNA strand, and four nucleic acid substrates for complementary strand synthesis (dATP, dCTP, dGTP, and dTTP) are added one by one in order into the reaction solution, and thereby a complementary strand is synthesized. Upon complementary strand synthesis, as the complementary DNA strand extends, pyrophosphate (PPi) is generated as a by-product. In the presence of an enzyme, pyrophosphate is converted to ATP, which in turn goes through the reaction in the presence of luciferin and luciferase to generate emission. By detecting this emission, the incorporation of the nucleic acid substrates for complementary strand synthesis in the DNA strand is confirmed, and the sequence information of the complementary strand, and consequently the sequence information of the target DNA strand will be elucidated. On the other hand, the nucleic acid substrates for complementary strand synthesis that have not been used in the reaction are promptly degraded by an enzyme such as apyrase so as not to interfere with subsequent reaction steps (for example, Patent document 2). Many apparatuses for this pyrosequencing employ chemiluminescent detection system, wherein a titer plate having 96 reaction cells (having a volume of 100 μl or less) is utilized as a reaction cell plate. In such apparatus, each of the four nucleic acid substrates for complementary strand synthesis (dATP, dCTP, dGTP, and dTTP) is contained in a separate reagent vessel and injected into the reaction cells one by one (for example, Patent document 3). That is, DNA, a primer, enzymes for synthesizing a complementary strand, chemiluminescent reagents, and the like are placed in advance in the reaction cells; a reagent dispenser comprises four nozzles; the nozzles or a titer plate is moved in the x-y directions as well as in the rotation direction; the air in the reagent vessels is pressurized; and thereby the reagents are dripped one by one from the tips of the nozzles, thus emission being detected.
Furthermore, a technology to provide a small size apparatus for the above pyrosequencing is disclosed (for example, Patent document 4). In this technology, a narrow tube is connected from each of dNTP vessels to the reaction section; it is suggested that compact and simple analysis is attainable by the method wherein four dNTPs are injected one by one by using these narrow tubes.
On the other hand, a luminescence detection apparatus utilizing a pressurized dispensing system for dispensing reagents is disclosed as a small size apparatus for measuring bioluminescence (for example, Patent document 5). In this technology, capillaries for dispensing are aligned with reaction cells one by one, and dispensing reagents is controlled by pressurization.
Moreover, in regarding to reagents that can be used for the pyrosequencing reaction, an example of a reaction system different from the technologies described above is disclosed (for example, Patent document 6). In this conventional technology, AMP and PPi are synthesized into ATP by using the reverse reaction of the enzyme, pyruvate, phosphate dikinase (PPDK), and AMP concentrations are measured.
Patent document 1: WO 98/13523
Patent document 2: WO 98/28440
Patent document 3: WO 00/56455
Patent document 4: JP-A-2001-258543
Patent document 5: JP-A-2004-12411
Patent document 6: JP-A-9-234099
It is believed that because the reaction mechanism used is simple, the pyrosequencing method is suitable for small size and inexpensive apparatuses. Four nucleic acid substrates for complementary strand synthesis are required for measurement, as described above, and hence these need to be measured accurately. In order to make an apparatus small and inexpensive, it is also essential to design to use a minute total amount of reagents.
In the conventional technology, there is a problem that an accurate reagent dispensing mechanism can not be small and inexpensive. For example, in order to make an apparatus small, dispensing about 0.1 to 0.2 μl needs to be performed within an error of 10% or less. However, conventionally, in the method of dripping reagents, which is said to be a simple method of dispensing reagents, for example, on dispensing 0.4 μl, a dispensing error of about 15% occurs, and on dispensing less than 0.4 μl, in many cases, dispensing is not possible due to surface tension of the liquid. Furthermore, another example to realize micro-dispensing is the Bubble Jet® technology, in general, used for inkjet printers, which has problems such that reagents are deteriorated by heating and that it is difficult to simplify replenishment and maintenance. Moreover, in the pressurized dispenser method using capillaries, which can realize simple, inexpensive, accurate dispensing, nevertheless, because the tip of the capillary is in contact with a sample solution in the reaction vessel, reagents may disadvantageously leak at the time the air is not pressurized.
Furthermore, four reagents need to be injected into a reaction vessel in a predetermined order. In the conventional nozzle method, there are problems that miniaturization is difficult and parallel arrangement is also difficult. That is, in a 96-well titer plate widely used in this field, 96 reaction vessels (holes) are placed with a pitch of 9 mm, but it is impossible to provide a plurality of nozzles with a pitch of 9 mm by the conventional technology. Therefore, the reagents are dispensed through a set of nozzles into multiple reaction vessels, so that the measurement efficiency is low as well as the horizontal mechanism movement tends to be large and expensive.
Furthermore, the mechanism that allows the dispensed substrates to admix with a sample in the reaction vessel efficiently is required. In order to realize a simple, small size, inexpensive apparatus, these problems should be solved.